The objective of this application is to request a Bruker BioSpin Corporation Elexsys E560 continuous-wave electron paramagnetic resonance (EPR) spectrometer with 10" electromagnet/12 kW power supply that will operate at X-band (9-10 GHz) and Q-band (34 GHz) microwave frequencies, and include the electron-nuclear double resonance (ENDOR, TRIPLE) accessories for X- and Q-band. Also requested are cryostats for operation at liquid helium and nitrogen temperature, to adjust to the different relaxation properties of the paramagnets, and a goniometer for single crystal studies. The new instrument would replace an aging EPR spectrometer system. The user group includes the PI, an experienced EPR spectroscopist, and three other Major Users and two Minor Users. The instrument will support the NIH-funded research of four users (5 R01 grants), the pending NIH-funded research of one user, and the preliminary studies of another. The proposed EPR/ENDOR projects include the following: (a) determination of the molecular mechanism of electrondeficient radical-mediated catalysis in coenzyme B12-dependent enzymes, (b) investigation of the emergent novel biological functions of iron-sulfur clusters in biotin synthase, pyruvate-formate lyase activating enzyme, and ferredoxin:thioredoxin reductase, in combination with Mossbauer spectroscopy, (c) the mechanism of biotransformation of aromatic hydrocarbons by toluene monooxygenases, by examining metal site structure in single crystals in combination with protein X-ray crystallography, (d) the structure and function of flavoenzymes, (e) determination of the role of divalent metals in the mechanism of fibrillogenesis in amyloid formation, and (f) characterization of the mechanism of radical-mediated DNA strand scission and histone modification by novel organometallic complexes. Operation at Q-band is justified by the need for enhanced resolution of both EPR and ENDOR spectra, and the dual X- and Q-band operation is essential for success of multi-frequency approaches to constrain spectral simulation analyses. The projects supported by the requested EPR spectrometer cover investigations of the wide range of recognized biological radical reactivity, from "programmed" essential radicals in enzymes, to deleterious effects of free radicals and metals, to the use of radical reactivity as a therapeutic tool. A sustained high impact of the EPR spectrometer on biomedicine is therefore anticipated.